1. Field of the Invention
The invention is generally related to forming glycosylated ligation products. In a particular embodiment, a thioacid is reacted with a compound which includes a glycosamine.
2. Background of Invention
The process of native chemical ligation (NCL)1 involves the chemoselective coupling of unprotected thioesters (Group A) (Group B) via an intramolecular S- to N-acyl transfer to give products of the type A-Cys-B, wherein Cys represents the amino acid residue cysteine.
NCL was a significant achievement because it enabled the synthesis of large peptides and proteins (“protein semisynthesis”) under very mild reaction conditions. A key feature of NCL is that it does not require the use of protecting groups and thus represents a particularly powerful approach to specifically modified proteins. The prototypical NCL process is characterized by the chemoselective coupling of “Group A”, an unprotected peptide thioester peptide1-Xaa-SR) and a “Group B” amino functionalized compound bearing a branched side chain that includes a removable thiol auxiliary, e.g. an unprotected cysteinyl peptide (H-Cys-peptide2). The resulting ligation product is A-Cys-B, where Cys represents the amino acid residue cysteine. If the reactants are peptides, the ligation product has the general structure peptide1-Xaa-Cys-peptide2.
The rate-determining step for NCL has been shown to be transthioesterification of the peptide thioester by the Cys thiol.2 Unfortunately, since an N-terminal Cys residue is required,3 and since the frequency of occurrence of Cys in proteins (1.82%) is low, NCL-based strategies are rather limited, e.g. to ligation at peptide linkages Xaa-Cys, where Xaa is preferably an unhindered amino acid. It follows that the incorporation of post-translationally modified or unnatural amino acids at the ligation site is not generally feasible with NCL.
The lack of generality and coupling efficiency represent significant gaps in the existing NCL technology repertoire.
Shao et al. (Chemistry & Biology 1994 (1) 4:231-234) describe aziridine containing peptides. However, the peptides were synthesized by conventional methods using protecting groups.
Rotstein et al. (Nature Protocols 2010 (5) 11: 1813-22) describe the synthesis of peptides that contain an aziridine ring which can be modified by ring opening. However, the method employs aziridine aldehydes, and the peptides are all macrocyclic and the products do not have a natural peptide backbone.
Weiss et al., (PNAS 1996. 93:10945-10948) describe the synthesis of an azirdine-containing peptide. However, the peptide is an arginine mimic and the aziridine is present on a side chain of the peptide.
Assem et al., (J. Am. Chem. Soc. 2010, 132, 10986-10987), describes chemoselective peptidomimetic ligation using thioacid peptides and aziridine templates. However, the peptide with the aziridine employs protecting groups, and the resulting peptides necessarily contain an SH group, unless removed by a step of desulfurization. Further, the linkage formed is not an alpha-peptide but a beta-peptide.
Galonic et al., (J. Am. Chem. Soc. 2005, 127, 7359-7369), describe aziridine-2-carboxylic acid-containing peptides. However, the synthesis method employs conventional protecting groups, and the aziridine containing peptides were reacted with thiols while they were still protected and attached to the solid phase
The covalent union of the carbohydrate and peptide domains of glycoproteins remains a formidable challenge. Recent synthetic approaches to N-linked glycoprotein constructs have generally utilized the native chemical ligation of glycopeptide segments, which are prepared by incorporating the glycosylated amino acids during a solid phase peptide synthesis. Syntheses of N-glycopeptides arising from the coupling of a glycosylamine with an aspartic acid residue embedded in a peptide to give the N-glycosylated peptide have also been reported. Inherent limitations of these protocols necessitate the masking of free amino groups (N-terminus, Lys sidechains) in the peptide, restricting their application to the middle and early stages of a projected glycoprotein synthesis. An aspartylation procedure that circumvents this chemoselectivity issue would permit the introduction of the glycan moiety at a later stage in the synthesis, thus making it more convergent.
Wang et al., (J. Am. Chem. Soc. 2011, 133, 1597-1602), describes the coupling of peptides containing a unique thioacid at the ω-aspartate carboxyl with glycosylamines to give N-linked glycopolypeptides. However, the reaction mechanism involves oxidation of the thioacid to give an active ester intermediate so that the N-terminal and sidechain amines must be protected.